Global impact and application of the anaerobic ammonium-oxidizing (anammox) bacteria.

In the anaerobic ammonium oxidation (anammox) process, ammonia is oxidized with nitrite as primary electron acceptor under strictly anoxic conditions. The reaction is catalysed by a specialized group of planctomycete-like bacteria. These anammox bacteria use a complex reaction mechanism involving hydrazine as an intermediate. The reactions are assumed to be carried out in a unique prokaryotic organelle, the anammoxosome. This organelle is surrounded by ladderane lipids, which make the organelle nearly impermeable to hydrazine and protons. The localization of the major anammox protein, hydrazine oxidoreductase, was determined via immunogold labelling to be inside the anammoxosome. The anammox bacteria have been detected in many marine and freshwater ecosystems and were estimated to contribute up to 50% of oceanic nitrogen loss. Furthermore, the anammox process is currently implemented in water treatment for the low-cost removal of ammonia from high-strength waste streams. Recent findings suggested that the anammox bacteria may also use organic acids to convert nitrate and nitrite into dinitrogen gas when ammonia is in short supply.

Biological removal of NOx from flue gas.

BioDeNOx is a novel integrated physico-chemical and biological process for the removal of nitrogen oxides (NOx) from flue gas. Due to the high temperature of flue gas the process is performed at a temperature between 50-55 degrees C. Flue gas containing CO2, O2, SO2 and NOx, is purged through Fe(II)EDTA2- containing liquid. The Fe(II)EDTA2- complex effectively binds the NOx; the bound NOx is converted into N2 in a complex reaction sequence. In this paper an overview of the potential microbial reactions in the BioDeNOx process is discussed. It is evident that though the process looks simple, due to the large number of parallel potential reactions and serial microbial conversions, it is much more complex. There is a need for a detailed investigation in order to properly understand and optimise the process.

Anaerobic ammonium oxidation by marine and freshwater planctomycete-like bacteria.

Recently, two fresh water species, " Candidatus Brocadia anammoxidans" and " Candidatus Kuenenia stuttgartiensis", and one marine species, " Candidatus Scalindua sorokinii", of planctomycete anammox bacteria have been identified. " Candidatus Scalindua sorokinii" was discovered in the Black Sea, and contributed substantially to the loss of fixed nitrogen. All three species contain a unique organelle--the anammoxosome--in their cytoplasm. The anammoxosome contains the hydrazine/hydroxylamine oxidoreductase enzyme, and is thus the site of anammox catabolism. The anammoxosome is surrounded by a very dense membrane composed almost exclusively of linearly concatenated cyclobutane-containing lipids. These so-called 'ladderanes' are connected to the glycerol moiety via both ester and ether bonds. In natural and man-made ecosystems, anammox bacteria can cooperate with aerobic ammonium-oxidising bacteria, which protect them from harmful oxygen, and provide the necessary nitrite. The cooperation of these two groups of ammonium-oxidising bacteria is the microbial basis for a sustainable one reactor system, CANON (completely autotrophic nitrogen-removal over nitrite) to remove ammonia from high strength wastewater.

CANON and Anammox in a gas-lift reactor.

Anoxic ammonium oxidation (Anammox) and Completely Autotrophic Nitrogen removal Over Nitrite (CANON) are new and promising microbial processes to remove ammonia from wastewaters characterized by a low content of organic materials. These two processes were investigated on their feasibility and performance in a gas-lift reactor. The Anammox as well as the CANON process could be maintained easily in a gas-lift reactor, and very high N-conversion rates were achieved. An N-removal rate of 8.9 kg N (m(3) reactor)(-1) day(-1) was achieved for the Anammox process in a gas-lift reactor. N-removal rates of up to 1.5 kg N (m(3) reactor)(-1) day(-1) were achieved when the CANON process was operated. This removal rate was 20 times higher compared to the removal rates achieved in the laboratory previously. Fluorescence in situ hybridization showed that the biomass consisted of bacteria reacting to NEU, a 16S rRNA targeted probe specific for halotolerant and halophilic Nitrosomonads, and of bacteria reacting to Amx820, specific for planctomycetes capable of Anammox.

Completely autotrophic nitrogen removal over nitrite in one single reactor.

The microbiology and the feasibility of a new, single-stage, reactor for completely autotrophic ammonia removal were investigated. The reactor was started anoxically after inoculation with biomass from a reactor performing anaerobic ammonia oxidation (Anammox). Subsequently, oxygen was supplied to the reactor and a nitrifying population developed. Oxygen was kept as the limiting factor. The development of a nitrifying population was monitored by Fluorescence In Situ Hybridization and off-line activity measurements. These methods also showed that during steady state, anaerobic ammonium-oxidizing bacteria remained present and active. In the reactor, no aerobic nitrite-oxidizers were detected. The denitrifying potential of the biomass was below the detection limit. Ammonia was mainly converted to N2 (85%) and the remainder (15%) was recovered as NO3-. N2O production was negligible (less than 0.1%). Addition of an external carbon source was not needed to realize the autotrophic denitrification to N2.

The biological sulfur cycle: novel opportunities for environmental biotechnology.

Although the study of sulfur cycle bacteria was already started around the 1890s by the famous microbiologists Winogradsky and Beijerinck, there are nowadays still many new discoveries to be made about the metabolic properties, phylogenetic position and ecological behaviour of bacteria that play a role in the biological sulfur cycle. The current interest of the scientific community in the biological sulfur cycle is very high, especially because of the many special organisms that have recently been discovered in the deep sea and other environments characterised by extreme conditions (such as high salt, low/high pH or temperature) and also in bioreactor environments. This paper highlights some of these new discoveries and relates them to environmental biotechnology. It is concluded that the micro-organisms from the sulfur cycle offer unique opportunities for sulfur pollution abatement and sulfur recovery.

Isolation and properties of obligately chemolithoautotrophic and extremely alkali-tolerant ammonia-oxidizing bacteria from Mongolian soda lakes.

Five mixed samples prepared from the surface sediments of 20 north-east Mongolian soda lakes with total salt contents from 5 to 360 g/l and pH values from 9.7 to 10.5 were used to enrich for alkaliphilic ammonia-oxidizing bacteria. Successful enrichments at pH 10 were achieved on carbonate mineral medium containing 0.6 M total Na(+) and < or =4 mM NH(4)Cl. Five isolates (ANs1-ANs5) of ammonia-oxidizing bacteria capable of growth at pH 10 were obtained from the colonies developed on bilayered gradient plates. The cells were motile and coccoid, with well-developed intracytoplasmic membranes (ICPM) and carboxysomes. At pH 10.0, ammonia was toxic for growth at concentrations higher than 5 mM NH(4)Cl. The bacteria were able to grow within the salinity range of 0.1-1.0 M of total Na+ (optimum 0.3 M). In media containing 0.3-0.6 M total Na(+), optimal growth in batch cultures occurred in the presence of a bicarbonate/carbonate buffer system within the pH range 8.5-9.5, with the highest pH limit at pH 10.5. At pH lower than 8.0, growth was slower, most probably due to decreasing free ammonia. The pH profile of the respiratory activity was broader, with limits at 6.5-7.0 and 11.0 and an optimum at 9.5-10.0. In pH-controlled, NH(3)-limited continuous culture, isolate ANs5 grew up to pH 11.3, which is the highest pH limit known for ammonia-oxidizing bacteria so far. This showed the existence of extremely alkali-tolerant ammonia-oxidizing bacteria in the soda lakes. Comparative 16S rDNA sequence analysis of the five isolates demonstrated that they possess identical 16S rDNA genes and that they are closely related to Nitrosomonas halophila (sequence similarity 99.3%), a member of the beta-subclass of the Proteobacteria. This affiliation was confirmed by comparative sequence analysis of the amoA gene, encoding the active-site subunit of the ammonia-monoxygenase, of one of the isolates. DNA-DNA hybridization data further supported that the soda lake isolates are very similar to each other and represent an alkali-tolerant subpopulation of N. halophila whose species description is herewith amended.

Dioxin contamination in food. Bayreuth, Germany, from September 28 to October 1, 2000.

Dioxin and PCB monitoring programs for food and feeding stuff in most countries of the world, including many European Countries are currently inadequate. Better control of food production lines and food processing procedures is needed to minimize entry of dioxin to the food chain and will help to avoid dioxin contamination accidents. This would also improve the ability to trace back a possible contamination to its source. European guidelines for monitoring programs should be established to ensure comparable and meaningful results. These guidelines should define the minimum requirements for the design of monitoring programs, analytical methods, and quality assurance. Though data from Northern Europe shows that the general population exposure to dioxin and PCB has decreased during the last ten years these compounds continue to be a risk of accidental contamination of the food chain. The most prominent recent example is the Belgian dioxin contamination of feeding stuff in 1999. The Belgian dioxin contamination was not detected due to dioxin monitoring programs but by their direct biological effects seen in animals. Four other cases of dioxin contamination have been detected in Europe since 1997 due to local monitoring programs. One of them (citrus pulp pellets 1998) was in a much larger scale than the Belgian dioxin contamination. The general population's exposure to dioxins and PCBs is still in the same range (1-4 pg WHO-TEQ/kg body weight and day) as the recently revised WHO tolerable daily intake (TDI). There is concern that short-term high level exposure to dioxins, furans, and PCB may cause biological effects on the human fetal development and further research is required. Further actions to control sources building on considerable advances already made in many countries may need to be supplemented by measures to prevent direct contamination of feeding stuff or food to reduce general population exposure further.

Denitrification at extremely high pH values by the alkaliphilic, obligately chemolithoautotrophic, sulfur-oxidizing bacterium Thioalkalivibrio denitrificans strain ALJD.

Thioalkalivibrio denitrificans is the first example of an alkaliphilic, obligately autotrophic, sulfur-oxidizing bacterium able to grow anaerobically by denitrification. It was isolated from a Kenyan soda lake with thiosulfate as electron donor and N2O as electron acceptor at pH 10. The bacterium can use nitrite and N2O, but not nitrate, as electron acceptors during anaerobic growth on reduced sulfur compounds. Nitrate is only utilized as nitrogen source. In batch culture at pH 10, rapid growth was observed on N2O as electron acceptor and thiosulfate as electron donor. Growth on nitrite was only possible after prolonged adaptation of the culture to increasing nitrite concentrations. In aerobic thiosulfate-limited chemostats, Thioalkalivibrio denitrificans strain ALJD was able to grow between pH values of 7.5 and 10.5 with an optimum at pH 9.0. Growth of the organism in continuous culture on N2O was more stable and faster than in aerobic cultures. The pH limit for growth on N2O was 10.6. In nitrite-limited chemostat culture, growth was possible on thiosulfate at pH 10. Despite the observed inhibition of N2O reduction by sulfide, the bacterium was able to grow in sulfide-limited continuous culture with N2O as electron acceptor at pH 10. The highest anaerobic growth rate with N2O in continuous culture at pH 10 was observed with polysulfide (S8(2-)) as electron donor. Polysulfide was also the best substrate for oxygen-respiring cells. Washed cells at pH 10 oxidized polysulfide to sulfate via elemental sulfur in the presence of N2O or O2. In the absence of the electron acceptors, elemental sulfur was slowly reduced which resulted in regeneration of polysulfide. Cells of strain ALJD grown under anoxic conditions contained a soluble cd1-like cytochrome and a cytochrome-aa3-like component in the membranes.

Microbial thiocyanate utilization under highly alkaline conditions.

Three kinds of alkaliphilic bacteria able to utilize thiocyanate (CNS-) at pH 10 were found in highly alkaline soda lake sediments and soda soils. The first group included obligate heterotrophs that utilized thiocyanate as a nitrogen source while growing at pH 10 with acetate as carbon and energy sources. Most of the heterotrophic strains were able to oxidize sulfide and thiosulfate to tetrathionate. The second group included obligately autotrophic sulfur-oxidizing alkaliphiles which utilized thiocyanate nitrogen during growth with thiosulfate as the energy source. Genetic analysis demonstrated that both the heterotrophic and autotrophic alkaliphiles that utilized thiocyanate as a nitrogen source were related to the previously described sulfur-oxidizing alkaliphiles belonging to the gamma subdivision of the division Proteobacteria (the Halomonas group for the heterotrophs and the genus Thioalkalivibrio for autotrophs). The third group included obligately autotrophic sulfur-oxidizing alkaliphilic bacteria able to utilize thiocyanate as a sole source of energy. These bacteria could be enriched on mineral medium with thiocyanate at pH 10. Growth with thiocyanate was usually much slower than growth with thiosulfate, although the biomass yield on thiocyanate was higher. Of the four strains isolated, the three vibrio-shaped strains were genetically closely related to the previously described sulfur-oxidizing alkaliphiles belonging to the genus Thioalkalivibrio. The rod-shaped isolate differed from the other isolates by its ability to accumulate large amounts of elemental sulfur inside its cells and by its ability to oxidize carbon disulfide. Despite its low DNA homology with and substantial phenotypic differences from the vibrio-shaped strains, this isolate also belonged to the genus Thioalkalivibrio according to a phylogenetic analysis. The heterotrophic and autotrophic alkaliphiles that grew with thiocyanate as an N source possessed a relatively high level of cyanase activity which converted cyanate (CNO-) to ammonia and CO2. On the other hand, cyanase activity either was absent or was present at very low levels in the autotrophic strains grown on thiocyanate as the sole energy and N source. As a result, large amounts of cyanate were found to accumulate in the media during utilization of thiocyanate at pH 10 in batch and thiocyanate-limited continuous cultures. This is a first direct proof of a "cyanate pathway" in pure cultures of thiocyanate-degrading bacteria. Since it is relatively stable under alkaline conditions, cyanate is likely to play a role as an N buffer that keeps the alkaliphilic bacteria safe from inhibition by free ammonia, which otherwise would reach toxic levels during dissimilatory degradation of thiocyanate.

The CANON system (Completely Autotrophic Nitrogen-removal Over Nitrite) under ammonium limitation: interaction and competition between three groups of bacteria.

The CANON system (Completely Autotrophic Nitrogen Removal Over Nitrite) can potentially remove ammonium from wastewater in a single, oxygen-limited treatment step. The usefulness of CANON as an industrial process will be determined by the ability of the system to recover from major disturbances in feed composition. The CANON process relies on the stable interaction between only two bacterial populations: Nitrosomonas-like aerobic and Planctomycete-like anaerobic ammonium oxidising bacteria. The effect of extended periods of ammonium limitation was investigated at the laboratory scale in two different reactor types (sequencing batch reactor and chemostat). The lower limit of effective and stable nitrogen removal to dinitrogen gas in the CANON system was 0.1 kg N m(-3) day(-1). At this loading rate, 92% of the total nitrogen was removed. After prolonged exposure (> 1 month) to influxes lower than this critical NH4+-influx, a third population of bacteria developed in the system and affected the CANON reaction stoichiometry, resulting in a temporary decrease in nitrogen removal from 92% to 57%. The third group of bacteria were identified by activity tests and qualititative FISH (Fluorescence In Situ Hybridisation) analysis to be nitrite-oxidising Nitrobacter and Nitrospira species. The changes caused by the NH4+-limitation were completely reversible, and the system re-established itself as soon as the ammonium limitation was removed. This study showed that CANON is a robust system for ammonium removal, enduring periods of up to one month of ammonium limitation without irreversible damage.

A new facultatively autotrophic hydrogen- and sulfur-oxidizing bacterium from an alkaline environment.

An alkaliphilic bacterium, strain AHO 1, was isolated from an enrichment culture with hydrogen at pH 10 inoculated with a composite sample of sediments from five highly alkaline soda lakes (Kenya). This bacterium is a gram-negative, nonmotile, rod-shaped, obligately aerobic, and facultatively autotrophic hydrogen-oxidizing organism. It was able to oxidize reduced sulfur compounds to sulfate during heterotrophic growth. It utilized a wide range of organic compounds as carbon and energy sources and grew mixotrophically with hydrogen and acetate. With sulfur compounds, mixotrophic growth was observed only in acetate-limited continuous culture. The normal pH range for autotrophic growth with hydrogen was pH 8.0-10.25, with a pH optimum at 9-9.5. Growth at pH values lower than 8.0 was extremely slow. Heterotrophic growth with acetate was optimal at pH 10.0. The hydrogen-oxidizing activity of whole cells was maximal at pH 9.0 and still substantial up to pH 11. NAD-dependent hydrogenase activity was found in the soluble fraction of the cell-free extract, but no methylene blue-dependent activity in either the soluble or membrane fractions was observed. On the basis of its pH profile, the soluble hydrogenase of strain AHO 1 was a typical pH-neutral enzyme. Phylogenetic analysis revealed that strain AHO 1 belongs to the alpha-3 subgroup of the Proteobacteria with a closest relation to a recently described alkaliphilic aerobic bacteriochlorophyll a-containing bacterium "Roseinatronobacter thiooxidans."

An obligate methylotrophic, methane-oxidizing Methylomicrobium species from a highly alkaline environment.

A new, obligately methylotrophic, methane-oxidizing bacterium, strain AMO 1, was isolated from a mixed sample of sediments from five highly alkaline soda lakes (Kenya). Based on its cell ultrastructure and high activity of the hexulose-6-phosphate synthase, the new isolate belongs to the type I methanotrophs. It differed, however, from the known neutrophilic methanotrophs by the ability to grow and oxidize methane at high pH values. The bacterium grew optimally with methane at pH 9-10. The oxidation of methane, methanol, and formaldehyde was optimal at pH 10, and cells were still active up to pH 11. AMO 1 was able to oxidize ammonia to nitrite at high pH. A maximal production of nitrite from ammonia in batch cultures at pH 10 was observed with 10% of CH4 in the gas phase when nitrate was present as nitrogen source. Washed cells of AMO 1 oxidized ammonia most actively at pH 10-10.5 in the presence of limiting amounts of methanol or CH4. The bacterium was also capable of oxidizing organic sulfur compounds at high pH. Washed cells grown with methane exhibited high activity of CS2 oxidation and low, but detectable, levels of DMS and DMDS oxidation. The GC content of AMO 1 was 50.9mol%. It showed only weak DNA homology with the previously described alkaliphilic methanotroph "Methylobacter alcaliphilus" strain 20Z and with the neutrophilic species of the genera Methylobacter and Methylomonas. According to the 16S rRNA gene sequence analysis, strain AMO 1 was most closely related to a neutrophilic methanotroph, Methylomicrobium pelagicum (98.2% sequence similarity), within the gamma-Proteobacteria.

Involvement of a novel hydroxylamine oxidoreductase in anaerobic ammonium oxidation.

In this study a novel hydroxylamine oxidoreductase (HAO) was purified and characterized from an anaerobic ammonium-oxidizing (Anammox) enrichment culture. The enzyme, which constituted about 9% of the protein mass in the soluble fraction of the cell extract, was able to oxidize hydroxylamine and hydrazine. When phenazine methosulfate and methylthiazolyltetrazolium bromide were used as electron acceptors, a V(max) [21 and 1.1 micromol min(-)(1) (mg of protein)(-)(1)] and K(m) (26 and 18 microM) for hydroxylamine and hydrazine were determined, respectively. The hydroxylamine oxidoreductase is a trimer and contains about 26 hemes per 183 kDa. As deduced from UV/vis spectra, hydroxylamine reduced more and different cytochromes than hydrazine. The dithionite-reduced spectrum showed an unusual 468 nm peak. Inhibition experiments with H(2)O(2) showed that hydroxylamine bound to this P-468 cytochrome, which is assumed to be the putative substrate binding site. Cyanide and hydrazine inhibited the oxidation of hydroxylamine. The amino acid sequences of several peptide fragments of HAO from Anammox showed a clear difference with the deduced amino acid sequence of HAO from the aerobic ammonia-oxidizing bacterium Nitrosomonas europaea. In EPR spectra of the Anammox HAO, two g-values (g(z)() = 2.37 and 2.42) were observed, which were not present in HAO of N. europaea.

Isolation and characterization of alkaliphilic, chemolithoautotrophic, sulphur-oxidizing bacteria.

Alkaliphilic sulphur-oxidizing bacteria were isolated from samples from alkaline environments including soda soil and soda lakes. Two isolates, currently known as strains AL 2 and AL 3, were characterized. They grew over a pH range 8.0-10.4 with an optimum at 9.5-9.8. Both strains could oxidize thiosulphate, sulphide, polysulphide, elemental sulphur and tetrathionate. Strain AL 3 more actively oxidized thiosulphate and sulphide, while isolate AL 2 had higher activity with elemental sulphur and tetrathionate. Isolate AL 2 was also able to oxidize trithionate. The pH optimum for thiosulphate and sulphide oxidation was between 9-10. Some activity remained at pH 11, but was negligible at pH 7. Metabolism of tetrathionate by isolate AL 2 involved initial anaerobic hydrolysis to form sulphur, thiosulphate and sulphate in a sequence similar to that in other colourless sulphur-oxidizing bacteria. Sulphate was produced by both strains. During batch growth on thiosulphate, elemental sulphur and sulphite transiently accumulated in cultures of isolates AL 2 and AL 3, respectively. At lower pH values, both strains accumulated sulphur during sulphide and thiosulphate oxidation. Both strains contained ribulose bisphosphate carboxylase. Thiosulphate oxidation in isolate AL 3 appeared to be sodium ion-dependent. Isolate AL 2 differed from AL 3 by its high GC mol % value (65.5 and 49.5, respectively), sulphur deposition in its periplasm, the absence of carboxysomes, lower sulphur-oxidizing capacity, growth kinetics (lower growth rate and higher growth yield) and cytochrome composition.

Missing lithotroph identified as new planctomycete.

With the increased use of chemical fertilizers in agriculture, many densely populated countries face environmental problems associated with high ammonia emissions. The process of anaerobic ammonia oxidation ('anammox') is one of the most innovative technological advances in the removal of ammonia nitrogen from waste water. This new process combines ammonia and nitrite directly into dinitrogen gas. Until now, bacteria capable of anaerobically oxidizing ammonia had never been found and were known as "lithotrophs missing from nature". Here we report the discovery of this missing lithotroph and its identification as a new, autotrophic member of the order Planctomycetales, one of the major distinct divisions of the Bacteria. The new planctomycete grows extremely slowly, dividing only once every two weeks. At present, it cannot be cultivated by conventional microbiological techniques. The identification of this bacterium as the one responsible for anaerobic oxidation of ammonia makes an important contribution to the problem of unculturability.

Key physiology of anaerobic ammonium oxidation.

The physiology of anaerobic ammonium oxidizing (anammox) aggregates grown in a sequencing batch reactor was investigated quantitatively. The physiological pH and temperature ranges were 6.7 to 8.3 and 20 to 43 degrees C, respectively. The affinity constants for the substrates ammonium and nitrite were each less than 0.1 mg of nitrogen per liter. The anammox process was completely inhibited by nitrite concentrations higher than 0.1 g of nitrogen per liter. Addition of trace amounts of either of the anammox intermediates (1. 4 mg of nitrogen per liter of hydrazine or 0.7 mg of nitrogen per liter of hydroxylamine) restored activity completely.

Hydroxylamine oxidation and subsequent nitrous oxide production by the heterotrophic ammonia oxidizer Alcaligenes faecalis.

Nitrous oxide (N2O), a greenhouse gas, is emitted during autotrophic and heterotrophic ammonia oxidation. This emission may result from either coupling to aerobic denitrification, or it may be formed in the oxidation of hydroxylamine (NH2OH) to nitrite (NO2(-). Therefore, the N2O production during NH2OH oxidation was studied with Alcaligenes faecalis strain TUD. Continuous cultures of A. faecalis showed increased N2O production when supplemented with increasing NH2OH concentrations. 15N-labeling experiments showed that this N2O production was not due to aerobic denitrification of NO2(-). Addition of 15N-labeled NH2OH indicated that N2O was a direct by-product of NH2OH oxidation, which was subsequently reduced to N2. These observations are sustained by the fact that NO2(-) production was low (0.23 mM maximum) and did not increase significantly with increasing NH2OH concentration in the feed. The NH2OH-oxidizing capacity increased with increasing NH2OH concentrations. The apparent Vmax and K(m) were 31 nmol min-1 mg dry weight-1 and 1.5 mM respectively. The culture did not increase its growth yield and was not able to use NH2OH as the sole N source. A non-haem hydroxylamine oxidoreductase was partially purified from A. faecalis strain TUD. The enzyme could only use K3Fe(CN)6 as an electron acceptor and reacted with antibodies raised against the hydroxylamine oxidoreductase of Thiosphaera pantotropha.

Isolation and characterization of a novel facultatively alkaliphilic Nitrobacter species, N. alkalicus sp. nov.

Five strains of lithotrophic, nitrite-oxidizing bacteria (AN1-AN5) were isolated from sediments of three soda lakes (Kunkur Steppe, Siberia; Crater Lake and Lake Nakuru, Kenya) and from a soda soil (Kunkur Steppe, Siberia) after enrichment at pH 10 with nitrite as sole electron source. Morphologically, the isolates resembled representatives of the genus Nitrobacter. However, they differed from recognized species of this genus by the presence of an additional S-layer in their cell wall and by their unique capacity to grow and oxidize nitrite under highly alkaline conditions. The influence of pH on growth of one of the strains (AN1) was investigated in detail by using nitrite-limited continuous cultivation. Under such conditions, strain AN1 was able to grow at a broad pH range from 6.5 to 10.2, with an optimum at 9.5. Cells grown at pH higher than 9 exhibited a clear shift in the optimal operation of the nitrite-oxidizing system towards the alkaline pH region with respect to both reaction rates and the affinity. Cells grown at neutral pH values behaved more like neutrophilic Nitrobacter species. These data demonstrated the remarkable potential of the new nitrite-oxidizing bacteria for adaptation to varying alkaline conditions. The 16S rRNA gene sequences of isolates AN1, AN2, and AN4 showed high similarity (> or = 99.8%) to each other, and to sequences of Nitrobacter strain R6 and of Nitrobacter winogradskyi. However, the DNA-DNA homology in hybridization studies was too low to consider these isolates as new strains. Therefore, the new isolates from the alkaline habitats are described as a new species of the genus Nitrobacter, N. alkalicus, on the basis of their substantial morphological, physiological, and genetic differences from the recognized neutrophilic representatives of this genus.